Genes and Biochemical Characterization of Three Novel Chlorophyllase Isozymes from Brassica oleracea
Genes and Biochemical Characterization of Three Novel Chlorophyllase Isozymes from Brassica oleracea
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DOI：
10.1021/jf1016384
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发表时间：
2010-08-11
影响因子：
6.1
通讯作者：
Shaw, Jei-Fu
中科院分区：
文献类型：
--
作者：
Lee, Guan-Chiun;Chepyshko, Hanna;Shaw, Jei-Fu
从青花菜（Brassica oleracea）小花cDNA库中克隆了3个叶绿素酶基因的全长cDNA（BoCLH 1，1140 bp; BoCLH 2，1104 bp; BoCLH 3，884 bp），并对其进行了鉴定。氨基酸序列分析表明，这3个BoCLH均含有一个高度保守的脂肪酶基序（GXSXG）。然而，只有BoCLH 3缺乏His残基，其是催化三联体（Ser-His-Asp）的组分。BoCLH 1和BoCLH 2的N-末端序列被预测为具有叶绿体的典型信号序列，而质膜靶向序列在BoCLH 3中被鉴定。BoCLH 1、2和3的预测分子量分别为34.7、35.3和23.5 kDa。在大肠杆菌中成功表达了重组BoCLH，并对其进行了生物学特性分析。重组BoCLH 3表现出非常低的叶绿素酶活性，可能是由于其不完全的催化三联体。BoCLH 1和BoCLH 2在pH稳定性和最适温度等生化特性上存在显著差异。动力学分析表明，BoCLH 1优选水解无镁叶绿素，而BoCLH 2水解叶绿素和无镁叶绿素在类似的水平。BoCLH 1和BoCLH 2的不同特性暗示它们在青花菜中可能具有不同的生理功能。重组BoCLH 2的催化三联体通过定点突变被鉴定为Ser 141，His 247和Asp 170。表明这3种叶绿素酶同工酶均为丝氨酸水解酶。
Three full length cDNAs (BoCLH1, 1140 bp; BoCLH2, 1104 bp; BoCLH3, 884 bp) encoding putative chlorophyllases were cloned from the cDNA pools of broccoli (Brassica oleracea) florets and characterized. The amino acid sequence analysis indicated that these three BoCLHs contained a highly conserved lipase motif (GXSXG). However, only BoCLH3 lacked the His residue which is the component of the catalytic triad (Ser-His-Asp). N-terminal sequences of BoCLH1 and BoCLH2 were predicted to have typical signal sequences for the chloroplast, whereas the plasma membrane-targeting sequence was identified in BoCLH3. The predicted molecular masses of BoCLH1, 2, and 3 were 34.7, 35.3, and 23.5 kDa, respectively. The recombinant BoCLHs were successfully expressed in Escherichia coli for the biochemical characterization. The recombinant BoCLH3 showed very low chlorophyllase activity possibly due to its incomplete catalytic triad. BoCLH1 and BoCLH2 showed significant differences in biochemical properties such as pH stability and temperature optimum. Kinetic analysis revealed that BoCLH1 preferably hydrolyzed Mg-free chlorophyll, while BoCLH2 hydrolyzed both chlorophyll and Mg-free chlorophyll at a similar level. Different characteristics between BoCLH1 and BoCLH2 implied that they may have different physiological functions in broccoli. The catalytic triad of recombinant BoCLH2 was identified as Ser141, His247, and Asp170 by site-directed mutagenesis. It suggested that the three broccoli chlorophyllase isozymes were serine hydrolases.